Bacterio-opsin is the sole protein found in the purple membrane of the halophilic archaebacterium, Halobacterium halobium. The complex of this protein with the chromophore, retinal, constitutes bacteriorhodopsin (bR) which functions as a light-driven proton pump to generate energy for the cell. Purple membrane synthesis is regulated by environmental factors such as light intensity and oxygen tension. Our discovery of two genes (brp and bac) associated with the expression of the bacterio-opsin (bop) gene has provided the first insight into a possible regulatory mechanism. The long term objectives of this proposal are: (i) to understand the regulation of bop gene expression in H. halobium, and (ii) to elucidate the function of bR at the structural level. One aim is to determine the functions of a cluster of genes flanking the bop gene and to define the role of light and oxygen in the regulation of these genes. Gene expression will be assayed under a variety of culture conditions which suppress or enhance purple membrane synthesis. Determination of mRNA levels expressed from these genes along with characterization of their protein products will assist in defining gene functions and regulatory mechanisms. Insights into the regulation of gene expression in the halophilic archaebacteria and the evolution of gene structure and function in all three cellular kingdoms will likely be revealed. A second aim is to establish a DNA transformation system for H. halobium. Halocin resistance and the bop gene will be developed for use as genetic markers. Shuttle vectors will be constructed and used as carriers for these markers and/or for genomic libraries from antibiotic or halocin resistant mutants. Once available, such a system can be used: (i) to augment investigations of the regulation of bop gene expression by complementation analysis and gene fusion, and (ii) to express site-directed mutations of the bop gene in the native organism. A third aim is to determine the specific amino acids of bacteriorhodopsin which are involved in transmembrane proton translocation and cation binding. Many individual site-directed mutations of the bop gene will be generated and expressed in E. coli until a DNA transformation system is available for H. halobium. Our collaborators, Prof. R. Stroud (UCSF) and Prof. D. Kliger (UCSC), will assess the function and structure of mutant bop proteins. Results obtained will contribute to an understanding of the basic processes of light/energy transduction and membrane biology.